Despite advances in cytokine-based therapeutics, such as neutralizing antibodies, new therapies, based on naturally occurring anti-inflammatory cytokines, have the potential to be effective. Interleukin-37 (IL-37), a member of the IL-1 family, has been a neglected cytokine without clear function. It has now become clear that IL-37 broadly suppresses innate inflammation and acquired immunity. The present application focuses on this cytokine and proposes to advance to potential therapeutic forms to treat human disease. Two of four aims of this proposal address outstanding issues related to the mechanism of action of IL-37. IL-37 being a dual function cytokine, similar to IL-1? and IL-33, translocatesto the nucleus but also binds to an extracellular receptor. Both exert anti-inflammatory properties. Therefore, we will generate a new strain of transgenic mice that lack the ability for IL-37 nuclear function and compare the responses to mice transgenic for the native form of human IL-37. IL-37 binds to the naturally occurring IL-18 binding protein (IL-18BP) and IL-18BP is in several clinical trials to treat auto-inflammatory diseases. We will assess the in vivo consequence of IL-18BP on the therapeutic benefit of recombinant IL-37 by using a newly developed strain of mice deficient in IL-18BP. IL-37 will be tested for its anti-inflammatory properties in IL-18BP deficien mice. Transgenic mice expressing human IL-37 will be treated with increasing doses of recombinant IL-18BP and determine how the concentrations of exogenous IL-18BP affect outcomes to inflammatory challenges. The third Aim directly addresses the question, what is the optimal form of IL-37 to develop as a therapeutic? Lacking a signal peptide, we will generate different N- and C-terminal forms of recombinant human IL-37 based on computerized molecular modeling, receptor binding assays and, most importantly, their anti-inflammatory and immunosuppressive properties using newly developed in vitro and in vivo assays. Once we have selected the sequence(s) of IL-37 for optimal anti-inflammatory properties, we will perform in vivo dosing studies in mice. In addition, we plan to produce a novel recombinant human IL-37 linked to the Fc domain of mouse IgG1 (IL-37:Fc) in order to increase in vivo efficacy of IL-37 in murine models of innate inflammation and immune-mediated diseases. The fourth Aim addresses polymorphisms in the IL-37 gene and how these affect the function of the cytokine. Using the novel technique of Molecular Internal Probe, we will be able to link common and rare mutations in the IL-37 gene to in vitro production in 850 subjects. Data from this Aim will contribute to the identification of persons who may benefit from IL-37 therapy. The overall goal of these studies is to advance the field of cytokine-based therapeutics, particularly of naturally occurring anti-inflammatory cytokines such as IL-37. The objectives of this proposal are to investigate how best to accomplish this goal.